This invention discloses a multi-layer three-dimensional single-molecule localization super-resolution imaging method and
system based on a
zoom lens, including step one: pre-experimental preparation, completing the calibration of the electrically adjustable lens
zoom parameters and obtaining the
astigmatism calibration curve; step two: fixing the sample, focusing, and activating the anti-drift function and
synchronous control program; step three: acquiring multi-layer three-dimensional single-molecule
scintillation images using a frame-by-frame scanning mode; and step four: performing three-dimensional localization and stitching reconstruction on all acquired single-molecule
fluorescence scintillation images. This invention overcomes the problems of slow scanning speed and mechanical
inertia associated with traditional axial scanning relying on the mechanical movement of the
microscope objective or stage. Furthermore, due to the rapid zooming and accurate resetting capabilities of ETL, a novel frame-by-frame scanning method can be used, solving the problem in traditional layer-by-layer scanning
modes where the focal plane of later scans is irradiated by
laser for a longer time than the focal plane of earlier scans, thus minimizing the
impact of
photobleaching on the
imaging quality of different
layers.